AimGlucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794.MethodsAC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2)]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F -cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14)] exenatide. Human GIP or [d-Ala(2)]GIP(1-42) were used for comparison.ResultsAC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2)]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2)]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174.ConclusionsThese studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

The design, synthesis and pharmacology of novel long-acting exenatide analogs for the treatment of metabolic diseases are described. These molecules display enhanced pharmacokinetic profile and potent glucoregulatory and weight lowering actions compared to native exenatide. [Leu14]exenatide-ABD is an 88 residue peptide amide incorporating an Albumin Binding Domain (ABD) scaffold. [Leu14]exenatide-ABP is a 53 residue peptide incorporating a short Albumin Binding Peptide (ABP). [Leu14]exenatide-ABD and [Leu14]exenatide-ABP exhibited nanomolar functional GLP-1 receptor potency and were metabolically stable in vitro in human plasma and in a pancreatic digestive enzyme mixture. Both molecules displayed picomolar and nanomolar binding association with albumin across multiple species and circulating half lives of 16 and 11 hours, respectively, post a single IV dose in rats. Unlike exenatide, both molecules elicited robust glucose lowering when injected 1 day prior to an oral glucose tolerance test, indicative of their extended duration of action. [Leu14]exenatide-ABD was compared to exenatide in a Lep ob/ob mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu14]exenatide-ABD displayed superior glucose lowering and weight loss in diabetic mice when compared to continuously infused exenatide at the same total weekly dose. A single oral administration of each molecule via an enteric coated capsule to cynomolgus monkeys showed superior pharmacokinetics for [Leu14]exenatide-ABD as compared to [Leu14]exenatide-ABP with detectable exposure longer than 14 days. These studies support the potential use of these novel long acting exenatide analogs with different routes of administration for the treatment of type 2 diabetes.

Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lepob/Lepob mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (HbA1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.

Introduction PYY is a 36-residue peptide first isolated from porcine intestine (Figure 1) [1,2]. Two endogenous forms of PYY, PYY(1-36) and the post-DPPIV [3,4] activated PYY(3-36), are released into the circulation following a meal [1,5]. PYY(3-36) appears to be the predominant secreted form. PYY has been known to inhibit gastric, [6] pancreatic and intestinal secretions [7]. PYY binds and activates at least four receptor subtypes (Y1, Y2, Y4 and Y5) [8-10] in rats and humans. These Y receptor subtypes display different patterns of affinity and activation for PYY, PYY(3-36) and synthetically modified PYY analogs. PYY(3-36) is a selective ligand for Y2 and Y5 receptors, implicated in food intake and feeding behavior, respectively [11]. In this study, PYY(3-36) analogs, where each residue of the natural sequence is replaced by L-alanine, and analogs with multiple alanine substitutions were synthesized. The three alanines at positions 7, 12 and 22 were replaced by a D-alanine. The affinity of each analog to the Y family of receptors and the ability of the analogs to decrease acute food intake in mouse are presented. Additionally, the efficacy of a single equimolar dose of PYY(3-36) and a subset of the analogs to reduce body weight in the chronic weight loss assay in the mouse is summarized.

475-P Inhibition of Long Chain Acyl Coenzyme A Synthetases During Fatty Acid Loading Induces Lipoapoptosis in Macrophages via Mitochondrial Dysfunction Saraswathi Viswanathan, Alyssa Hasty regulation of Gene expression 169-P Assessing Gene-Treatment Interactions at the FTO and INSIG2 Loci on Obesity-Related Traits in the Diabetes Prevention Program Paul Franks 170-P High-Fat Meal Induced Changes in the DuodenumMucosa Transcriptome Mayumi Yoshioka, Carl Bolduc, Vincent Raymond, Jonny St-Amand 171-P Acute Molecular Mechanisms Responsive to Feeding and Meal Constitution in Visceral Adipose Tissue Jonny St-Amand, Carl Bolduc, Mayumi Yoshioka, Sheng-Xiang Lin, Vincent Raymond 172-P DGAT, A Gene Associated with Triacylglycerol Synthesis, is Differentially Expressed in Tissues Associated with Metabolic Syndrome Michele Millham, William Zavadoski, Judith Treadway, Karen Houseknecht, Michael Gibbs

Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

Hepatitis C, which is caused by HCV, affects about 2% of the world’s population and is responsible for more than 50 million cases of hepatocellular carcinomas worldwide. HCV uses hepacivirin, a serine protease at the N-terminal third of the protein product of its NS3 gene, to process its NS4a-NS4b-NS5a-NS5b polyprotein. In addition, hepacivirin cleaves internally the NS3-NS4a site, releasing its cofactor NS4a. Inhibition of hepacivirin’s activity is one strategy for developing drugs to treat hepatitis C. Methods to measure the activity and inhibition of hepacivirin have included the use of polyproteins, peptides, and depsipeptides as substrates in PAGE, ELISA, BIAcore, and RP-HPLC-based assays [1-5]. More preferable are continuous chromogenic and fluorogenic assays, which use commercially available peptide-pNAs and peptide-AMCs, respectively. Additionally, an assay using peptide-p-phenylazophenyl ester as substrate has been developed [6], which is limiting due to a requirement for low pH and inherent high background and stability problems. Most recently, a continuous assay based on fluorescence resonance energy transfer (FRET) depsipeptide substrates has been presented [7]. Using the same FRET principle, we designed depsipeptide substrates for greater ease of chemical synthesis, lower molecular weight, and enhancing assay sensitivity with lower background, while maintaining the advantages of compatibility and stability at physiologic pH.

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[reaction: see text] The Passerini reaction of N-protected amino aldehydes, isonitriles, and TFA using pyridine-type bases proceeds under mild conditions and directly affords alpha-hydroxy-beta-amino amide derivatives in moderate to high yields. These adducts are readily hydrolyzed to alpha-hydroxy-beta-amino carboxylic acids. Application of these key intermediates to concise syntheses of P(1)-alpha-ketoamide protease inhibitors is illustrated.

Judicious combination of P-region sequences of highly potent anticoagulant proteins including NAP5, NAP6, Ecotin, and Antistasin with SAR from small molecule FXa inhibitors led to a series of chimeric inhibitors of formula 1a–j. We report herein the design, synthesis, and biological activity of this novel family of FXa inhibitors that express both high in vitro potency and superb selectivity against related serine proteases.